Use of indole-3-acetic acid derivatives in medicine

ABSTRACT

Compounds of formula (I), or physiologically functional derivatives thereof, wherein: R 1 , R 2 , R 3  and R′ 3  are independently selected from II or lower alkyl; and R 4 , R 5 , R 6  and R 7  are independently selected from H, electron withdrawing groups (such as F, Cl, Br, I, OCF 3 , carboxyl groups, acetal groups, electron deficient aryl groups), lower alkyl groups lower alkoxy groups, aryl groups or aryloxy groups, wherein it least one of R 4 , R 5 , R 6 , and R 7  is selected from an electron withdrawing group, may be used in methods of therapy, particular in treating neoplastic diseases in methods of GDEPT, ADPET, PDEPT and PDT

This invention relates to derivatives of indole-3-acetic acid (IAA) andtheir use as pharmaceuticals, in particular for treating neoplasticdisease.Indole-3-acetic acid:

is a naturally-occurring plant growth phytohormone which has beenextensively studied. As long ago as 1956, its effects in humans werestudied, and it was shown that single doses of 0.1 g/kg were non-toxic(Mirsky A and Diengott D, Hypoglycemic action of indole-3-acetic acid bymouth in patients with diabetes mellitus, Proc. Soc. Exp. Biol. Med. 93:109-110, 1956). In 1964, it was found that its photo-oxidation productsacted as growth inhibitors of microorganisms (Still C, Fukuyama T andMoyed H, Inhibitory Oxidation Products of Indole-3-acetic acid, J.Biological Chemistry, 240, 6, 2612-2618, 1964).

More recently, there has been interest in using indole-3-acetic acid asa prodrug in combination with horseradish peroxidase (HRP). IAA isviewed as a suitable prodrug as it is non-toxic in its stable form, itis not oxidised to any substantial extent by the human body's naturalperoxidases, but it is readily oxidised by HRP. HRP, and otherperoxidases, are enzymes which can be delivered to the desired site ofactivity by methods discussed below, such as, ADEPT and GDEPT.

For example, the enzyme may be linked to a monoclonal antibody so thatit can bind to an extra-cellular tumour-associated antigen. Thisapproach to cancer therapy, often referred to as “antibody directedenzyme/prodrug therapy” (ADEPT) is disclosed in WO88/07378, and iscurrently in Phase II clinical trials.

A variation on ADEPT is “polymer directed enzyme/prodrug therapy”(PDEPT), in which the compound to be targeted, for example the enzyme,is bound to a water soluble, biocompatible and non-immunogenic polymer.This polymer localises in the tumour due to the blood vessels in tumoursbeing leaky, allowing the polymer-bound molecules to enter theextra-cellular spaces in tumours, which does not occur very readily innormal tissues. Clearance from the tumour is slow due to lack oflymphatic drainage, allowing the localised enzyme-polymer to activatethe prodrug. PDEPT is described in more detail in “The Role of PolymerConjugates in the Diagnosis and Treatment of Cancer”, Duncan R et al,STP Pharma Sciences 6:237-263, 1996), and has recently completed Phase Itrials in administering cytotoxic drugs (e.g. doxorubicin) to tumours(Vasey P A et al, Phase I clinical and pharmacokinetic study of PK1[N-(2-hydroxypropyl) methacrylamide copolymer doxorubicin]: first memberof a new class of chemotherapeutic agents-drug-polymer conjugates, Clin.Cancer Res 5:83-94, 1999).

A further therapeutic approach termed “virus-directed enzyme prodrugtherapy” (VDEPT) is where tumour cells are targeted with a viral vectorcarrying a gene encoding the relevant enzyme. The gene may betranscriptionally regulated by tissue specific promoter or enhancersequences. The viral vector enters tumour cells and expresses theenzyme, thereby converting the prodrug into the active drug within thetumour cells (Huber et al., Proc. Natl. Acad. Sci. USA (1991) 88, 8039).

Alternatively, non-viral methods for the delivery of genes have beenused. Such methods include calcium phosphate co-precipitation,microinjection, liposomes and derivatives, electroporation, direct DNAuptake, and receptor-mediated DNA transfer. These are reviewed in Morgan& French, Annu. Rev. Biochem., 1993, 62;191. The term “GDEPT”(gene-directed enzyme prodrug therapy) is used to include both viral andnon-viral delivery systems. GDEPT is also currently in clinical trials.

Photodynamic therapy (PDT) involves the use of light to activatemolecules in order to produce toxic species. The majority of current andproposed technique's use singlet oxygen as the toxic species derivedfrom the reaction of a photosensitizer with cellular oxygen. Thetechnique's main drawback is that it does not work in anoxic tumours andthat the photosensitizers are not readily excreted by the body, andtherefore patients treated with PDT remain sensitive to light for aconsiderable period after treatment.

The mechanism of action of HRP on IAA is proposed to be as follows(Peroxidase-catalysed effects of Indole-3-acetic acid and analogues onlipid membranes, DNA, and mammalian cells in vitro, Folkes L et al,Biochemical Pharmacology, 57, 375-382, 1999):

In the diagram above, Nu refers to a cellular nucleophilic centre foundin, e.g. DNA or proteins. In the absence of oxygen, the skatole radicalmay react with biomolecules.

Unusually, IAA is oxidised by HRP without requiring hydrogen peroxide.

The exact nature of the cytotoxic action of the oxidised derivatives ofIAA is not known, although various possible mechanisms have beensuggested.

Recent work by the inventors has shown that when the aromatic ring ofIAA is substituted by electron donating substituents, such as methoxy,the rate of oxidation by HRP is increased, although cytotoxicity falls.

Surprisingly, the inventors have now found that when the aromatic ringof IAA is substituted by at least one electron withdrawing group, thecytotoxicity of the compounds, when activated by peroxidase, increases.This is particularly surprising in view of the slower reaction betweensuch substituted-IAAs and peroxidase.

Accordingly, a first aspect of the present invention provides the use ofcompounds of formula (I), or physiologically functional derivativesthereof, in a method of therapy:

wherein:R₁, R₂, R₃ and R′₃ are independently selected from H or lower alkyl; andR₄, R₅, R₆ and R₇ are independently selected from H, electronwithdrawing groups (such as F, Cl, Br, I, OCF₃, carboxyl groups, acetalgroups, electron deficient aryl groups), lower alkyl groups, loweralkoxy groups, aryl groups or aryloxy groups, wherein at least one ofR₄, R₅, R₆ and R₇ is selected from an electron withdrawing group.

‘Electron-withdrawing’ groups are those groups which reduce the electrondensity in other parts of the molecule. Those groups suitable for use inthe present invention include F, Cl, Br, I, OCF₃, carboxyl groups,acetal groups and electron deficient aryl groups. The preferred electronwithdrawing groups are F, Cl, Br and I, of which F, Cl and Br are mostpreferred.

‘Lower alkyl’ in this application means a group having from 1 to 7carbon atoms, which may be aliphatic or alicyclic, or a combinationthereof and which may be saturated, partially unsaturated, or fullyunsaturated. This group may bear one or more substituents, selected fromhalo (i.e. F, Cl, Br, I, preferably F, Cl, Br), carboxyl, acetal, aryl,aryloxy and alkoxy.

Examples of saturated linear lower alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, n-butyl, and n-pentyl (amyl).

Examples of saturated branched lower alkyl groups include, but are notlimited to, iso-propyl, iso-butyl, sec-butyl, tert-butyl, andneo-pentyl.

Examples of saturated alicyclic (carbocyclic) lower alkyl groups (alsoreferred to as “C₃₋₇ cycloalkyl” groups) include, but are not limitedto, unsubstituted groups such as cyclopropyl, cyclobutyl, cyclopentyl,and cyclohexyl, as well as substituted groups (e.g., groups whichcomprise such groups), such as methylcyclopropyl, dimethylcyclopropyl,methylcyclobutyl, dimethylcyclobutyl, methycyclopentyl,dimethycyclopentyl, methylcyclohexyl, dimethylcyclohexyl,cyclopropylmethyl and cyclohexylmethyl.

Examples of unsaturated lower alkyl groups which have one or morecarbon-carbon double bonds (also referred to as “C₂₋₇ alkenyl” groups)include, but are not limited to, ethenyl (vinyl, —CH═CH₂), 2-propenyl(allyl, —CH—CH═CH₂), isopropenyl (—C(CH₃)═CH₂), butenyl, pentenyl, andhexenyl.

Examples of unsaturated lower alkyl groups which have one or morecarbon-carbon triple bonds (also referred to as “C₂₋₇ alkynyl” groups)include, but are not limited to, ethynyl (ethinyl) and 2-propynyl(propargyl).

Examples of unsaturated alicyclic (carbocyclic) lower alkyl groups whichhave one or more carbon-carbon double bonds (also referred to as “C₃“cycloalkenyl” groups) include, but are not limited to, unsubstitutedgroups such as cyclopropenyl, cyclobutenyl, cyclopentenyl, andcyclohexenyl, as well as substituted groups (e.g. groups which comprisesuch groups) such as cyclopropenylmethyl and cyclohexenylmethyl.

‘Carboxyl’ in this application means a group of structure —C(═O)—, andincludes carboxylates, acyl groups, amides and acyl halides.

‘Carboxylates’ means groups of structure —C(═O)OR, where R is H or acarboxyl substituent, for example, a lower alkyl group or a C₅₋₂₀ arylgroup, preferably a lower alkyl group. Examples of carboxyl groupsinclude, but are not limited to —C(═O)OH, —C(═O)OCH₃, —C(═O)OCH2CH₃,—C(═O)OC(CH₃)₃, and —C(═O)OPh.

‘Acyl’ in this application means a group of structure —C(═O)R, where Ris H or an acyl substituent, for example, a lower alkyl group or a C₅₋₂₀aryl group, preferably a lower alkyl group. Examples of acyl groupsinclude, but are not limited to, —C(═O)H (formyl), —C(═O)CH₃ (acetyl),—C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃ (butyryl), and —C(═O)Ph(benzoyl, phenone).

‘Amides’ in this application means groups of structure —C(═O)NR₁R₂,wherein R¹ and R² are independently amino substituents, for example,hydrogen, a lower alkyl group (also referred to as C₁₋₇ alkylamino ordi-C₁₋₇ alkylamino), or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkylgroup. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)NH(CH₃)₂₁—C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

‘Acyilhalide’ means groups of structure —C(═O)X, wherein X is —F, —Cl,—Br, or —I, preferably —Cl, —Br, or —I.

‘Acetal’ in this application means a structure —C(OR³)(OR⁴)—, where thethird substituent is as for a carbonyl group (defined above). R³ and R⁴are selected from lower alkyl groups, or may together form a divalentalkyl group.

‘Alkoxy’ in this application means a group of structure —OR, wherein Ris an optionally substituted lower alkyl group, wherein the substituentmay include halo (F, CL, Br, I) and aryl. Examples of alkoxy groupsinclude, but are not limited to, —OCH₃ (methoxy), —OCH₂CH₃ (ethoxy),—OC(CH₃)₃ (tert-butoxy), —OBn (benzyloxy), and —OCH₂F (fluoromethoxy).

‘Aryl’ in this application means a monovalent moiety obtained byremoving a hydrogen atom from a ring atom of a C₅₋₂₀ aromatic compound(also known as a C₅₋₂₀ aryl group), said compound having one ring, ortwo or more rings (e.g. fused), and having from 5 to 20 ring atoms.Preferably, each ring has from 5 to 7 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”, inwhich case the group may conveniently be referred to as a “C₅₋₂₀carboaryl” group.

Examples of aryl groups which do not have ring heteroatoms (i.e. C₅₋₂₀carboaryl groups) include, but are not limited to, those derived frombenzene (i.e. phenyl), naphthalene, anthracene, phenanthrene, andpyrene.

Alternatively, the ring atoms may include one or more heteroatoms,including but not limited to oxygen, nitrogen, and sulfur, as in“heteroaryl groups.” In this case, the group may conveniently bereferred to as a “C₅₋₂₀ heteroaryl” group, wherein “C₅₋₂₀” denotes ringatoms, whether carbon atoms or heteroatoms. Preferably, each ring hasfrom 5 to 7 ring atoms, of which from 0 to 4 are ring heteroatoms.

Examples of C₅₋₂₀ heteroaryl groups include, but are not limited to, C₅heteroaryl groups derived from furan (oxole), thiophene (thiole),pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole),triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, andoxatriazole; and C₆ heteroaryl groups derived from isoxazine, pyridine(azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g.cytosine, thymine, uracil), pyrazine (1,4-diazine), triazine, tetrazole,and oxadiazole (furazan).

Examples of C₅₋₂₀ heterocyclic groups (including C₅₋₂₀ heteroarylgroups) which comprise fused rings, include, but are not limited to,those derived from quinoline, isoquinoline, purine (e.g., adenine,guanine), benzimidazole, carbazole, fluorene, phenoxathiin, benzofuran,indole, isoindole, quinoxaline, phenazine, phenoxazine, xanthene,acridine, and phenothiazine.

‘Electron deficient aryl groups’ in this application means an aryl groupwhich is electron withdrawing, and includes heteroaryl groups. Anexample of an electron deficient aryl group is para-chlorophenyl.

‘Aryloxy’ in this application means a group of structure —OR, wherein Ris an aryl group. An examples of an aryloxy group is —OPh (phenoxy).

Physiologically functional derivatives of prodrugs include salts, amidesand esters. Esters include carboxylic acid esters in which thenon-carbonyl moiety of the ester grouping is selected from straight orbranched chain C₁₋₆ alkyl (e.g. methyl, n-propyl, n-butyl or t-butyl);or C₃₋₆ cyclic alkyl (e.g. cyclohexyl). Salts include physiologicallyacceptable base salt, e.g. derived from an appropriate base, such asalkali metal (e.g. sodium, alkaline earth metal (e.g. magnesium) salts,ammonium and NR₁₄ (where R” is C₁₋₄ alkyl) salts. Other salts includeacid addition salts, including the hydrochloride and acetate salts.Amides include non-substituted and mono and di substituted derivatives.Such derivatives may be prepared by techniques known per se in the artof pharmacy.

It is preferred that the balance, or overall effect, of the substituentsR₄, R₅, R₆ and R₇ is electron withdrawing.

The following preferences for the groups R₁, R₂, R₃, R′₃, R₄, R₅, R₆ andR₇ may be independent of each other or may be in any combination witheach other.

R₁ and R₂ are preferably selected from H or optionally substitutedsaturated lower alkyl groups, more preferably optionally substitutedsaturated linear lower alkyl groups, more particularly methyl or ethyl.The most preferred group for R₁ and R₂ is H.

If the lower alkyl group of R₁ is substituted, the substituent ispreferably one which aids solubility of the whole compound, such asmorpholino or piperazinyl.

R′₃ is preferably H. R₃ is preferably selected from H or optionallysubstituted saturated lower alkyl groups, more preferably optionallysubstituted saturated linear lower alkyl groups, more particularlymethyl or ethyl. The most preferred group for R₃ is H, so that incombination R₃ and R′₃ are both H.

It is preferred that one or two, more preferably one, of R₄, R₅, R₆ andR₇ are independently selected from electron withdrawing groups.

If one or more of R₄, R₅, R₆ and R₇ are not H or an electron withdrawinggroups, they are preferably selected from optionally substitutedsaturated lower alkyl groups, more preferably optionally substitutedsaturated linear lower alkyl group and most preferably unsubstitutedlinear lower alkyl groups, more particularly methyl or ethyl.

It is most preferred that one of R₄, R₅, R₆ and R₇ is an electronwithdrawing group and the rest are H, and in particular that R₅ is theelectron withdrawing group.

One preferred aspect of the invention is the compounds of formula (I)which exhibit a greater cytotoxic effect (i.e. leave less survivingcells when tested under identical conditions) on a neoplastic cell linein vitro than indole-3-acetic acid. Preferably the cell line is hamsterlung fibroblast V79 from the European Tissue Culture Collection.

A second aspect of the present invention provides the use of a compoundof formula (I) as defined in the first aspect of the invention in themanufacture of a medicament for treating neoplastic disease.

A third aspect of the present invention provides the use of compounds offormula (I) as defined in the first aspect of the invention inconjunction with a conjugated peroxidase enzyme (for example andpreferably horseradish peroxidase) in methods of ADEPT and PDEPTtherapy, or in conjunction with a vector encoding and capable ofexpressing a peroxidase enzyme (for example and preferably horseradishperoxidase) in a tumour cell in a method of GDEPT. The drug produced bythe action of the peroxidase enzyme on the compounds of formula (I) maybe used for the selective killing of oxic and hypoxic tumour cells inmethods of treatments of cancers, for example leukaemias andparticularly solid cancers including breast, bowel, liver, head andneck, and lung tumours, including small cell carcinoma.

A fourth aspect of the present invention provides the use of compoundsof formula (I) as defined in the first aspect of the invention inconjunction with a photosensitizer (e.g. porphyrins, phenothiazines(methylene blue, toluidine blue, thionine), rose bengal, hypericin,phthalocyanines) in methods of photodynamic therapy (PDT).

The preferred embodiments of the second, third and fourth aspects of thepresent invention may relate to different subsets of compounds offormula (I).

The invention also provides pharmaceutical compositions comprising acompound of formula (I) as defined in the first aspect of the inventiontogether with a pharmaceutically acceptable carrier or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the variation in the surviving fraction of V79 cellsagainst varying concentration of a compound of formula (I),5-fluoro-indole-3-acetic acid, following 5-hours incubation, with orwithout HRP (1.2 μg/L).

FIG. 2 shows the variation in the surviving fraction of V79 cellsagainst the varying concentration of HRP, following 5 hours incubation,with 50 μM of the same compound of formula (I) as in FIG. 1,5-fluoro-indole-3-acetic acid.

FIG. 3 shows the variation in the surviving fraction of MCF7 and HT29cells when incubated for varying lengths of time with 100 μM of the samecompound of formula (I) as in FIGS. 1 and 2, 5-fluoro-indole-3-aceticacid and with or without HRP (1.2 μg/L).

FIG. 4 a shows the variation in the surviving fraction of V79 cellsfollowing 1 hour incubation against varying photolysis time of the cellsin conjunction with methylene blue, optionally in the presence ofcompounds of formula (I), 5-fluoro-indole-3-acetic acid or5-bromo-indole-3-acetic acid.

FIG. 4 b is similar to FIG. 4 a, except that thionine was used in placeof methylene blue.

FIG. 4 c is similar to FIG. 4 a, except that toluidine blue was used inplace of methylene blue.

FIG. 4 d is similar to FIG. 4 a, except that rose bengal was used inplace of methylene blue.

FIG. 5 shows the variation in the surviving fraction of transfected andcontrol T24 cells after 24 hours incubation against varyingconcentrations of compounds of formula (I), 5-fluoro-indole-3-aceticacid or 5-bromo-indole-3-acetic acid.

FIG. 6 shows the variation in concentration of 5-fluero-indole-3-aceticacid in plasma and tissues following intra-peritoneal injection intomice.

Synthesis Routes

Compounds of formula (I) may be made by various routes, some of whichare outlined below.

Fischer Indole Synthesis

The starting materials for this method are the correspondingphenylhydrazines, which are available commercially or can be synthesisedfrom the corresponding aniline. (KC Engvild, Acta Chem. Scand. B, 1977,31, 338-339; SW Fox, MW Bullock, J. Am. Chem. Soc., 1951, 73,2756-2759). The overall synthetic method is shown in scheme 1.

Side Chain Introduction

If the parent indole is available (e.g. 7-chloroindole) or synthesisedby one of the routes below, the compound of formula (I) can besynthesised by introduction of the acetate side chain onto the parentindole, using the reagents shown in Scheme 2. (RD Dillard et al., J.Med. Chem, 1996, 39, 5119-5136)

Halogenated Compounds

The parent indoles of compounds in which at least one of R₄, R₅, R₆, andR₇ are halogens can in particular be prepared by the Leimgrüber-Batchomethod, see Scheme 3 (AD Batcho, W Leimgrüber, Org. Synth., 1985, 63,214-225) or the Bartoli method, see Scheme 4 (G Bartoli, G Palmieri,Tet. Lett., 1989, 30, 2129-2132).

Aryl-substituted compounds

Indoles where one or more of R₄ to R₇ is an aryl group can be made bySuzuki coupling of the appropriate arylboronic acid with thecorresponding bromoindole, using a variation on the method employed byCarrera (GM Carrera, GS Sheppard, Synlett, 1994, 93-94) This is outlinedin Scheme 5.

R₁ can be introduced at any appropriate stage in the above synthesisroutes by the deprotonation of the indole nitrogen by a suitable base(for example, sodium hydride, sodium carbonate), followed by reactionwith an appropriate electrophile, preferably in the form RX, where X isa halogen (for example, iodomethane).

R₃ may be introduced by reaction of the substituted IAA (R₃=H) with thebase LDA, followed by an electrophile (AH Katz et al, Journal ofMedicinal Chemistry, 1988, 31, 1244-50). Alternatively, the desired sidechain could be introduced onto the parent indole, by for example, thereaction of the parent indole with sodium hydroxide, ethanol andlactonitrile to give the α-methyl IAA (Erdtmann et al, Acta ChemicaScandinavia., 1954, 8, 119-123).

GDEPT

Vector Systems

In general, the vector for use in GDEPT therapies may be any suitableDNA or RNA vectors.

Suitable non-viral vectors include cationic liposomes and polymers.Suitable viral vectors include those which are based upon a retrovirus.Such vectors are widely available in the art. Huber et al. (ibid) reportthe use of amphotropic retroviruses for the transformation of hepatoma,breast, colon or skin cells. Culver et al. (Science (1992) 256;1550-1552) also describe the use of retroviral vectors in GDEPT. Suchvectors or vectors derived from them may also be used. Otherretroviruses may also be used to make vectors suitable for use in thepresent invention. Such retroviruses include Rous sarcoma virus (RSV).

Englehardt et al. (Nature Genetics (1993) 4; 27-34) describe the use ofadenovirus-based vectors in the delivery of the cystic fibrosistransmembrane conductance product (CFTR) into cells, and suchadenovirus-based vectors may also be used. Vectors utilising adenoviruspromoter and other control sequences may be of particular use indelivering a system according to the invention to cells in the lung, andhence useful in treating lung tumours.

Other vector systems including vectors based on the Molony murineleukaemia virus are known (Ram, Z et al., Cancer Research (1993) 53;83-88; Dalton & Treisman, Cell (1992) 68; 597-612). These vectorscontain the Murine Leukaemia virus (MLV) enhancer cloned upstream at aβ-globin minimal promoter. The β-globin 5′ untranslated region up to theinitiation codon ATG is supplied to direct efficient translation of theenzyme.

Suitable promoters which may be used in vectors described above, includeMLV, (cytomegalovirus) CMV, RSV and adenovirus promoters. Preferredadenovirus promoters are the adenovirus early gene promoters. Strongmammalian promoters may also be suitable. An example of such a promoteris the EF-1α promoter which may be obtained by reference to Mizushimaand Nagata ((1990), Nucl. Acids Res., 18; 5322). Variants of suchpromoters retaining substantially similar transcriptional activities mayalso be used.

Other suitable promoters include tissue specific promoters, andpromoters activated by small molecules, hypoxia or X-rays. The use ofhypoxia regulated gene expression for gene therapy is described inEP-A-0745131.

HRP is the enzyme of choice for the activation of compounds of formula(I). Other suitable peroxidases include tobacco peroxidase, peanutperoxidase, lignin peroxidase, ascorbate peroxidase, bacterialcatalase-peroxidases, yeast cytochrome c peroxidase and Coprinuscinereus (Arthomyces ramosus, Coprinus macrorhizus) peroxidase.Synthetic peroxidases, e.g. oxoiron(IV)tetra(N-methylpyridyl)porphyrinsor microperoxidases may also be used. Microperoxidases may beadvantageous as they are small proteins, and thus less likely to causeimmunological problems. The enzymes may be modified by standardrecombinant DNA techniques, e.g. by cloning the enzyme, determining itsgene sequence and altering the gene sequence by methods such as fusion,truncation, substitution, deletion or insertion of sequences for exampleby site-directed mutagenesis. Reference may be made to “MolecularCloning” by Sambrook et al. (1989, Cold Spring Harbor) for discussion ofstandard recombinant DNA techniques. The modification made may be anywhich still leaves the enzyme with the ability to catalyse the formationof the radical cation in compounds of formula (I), but alters otherproperties of the enzyme, for example its rate of reaction, selectivityor immunological properties.

In addition, small truncations in the N- and/or C-terminal sequence mayoccur as a result of the manipulations required to produce a vector inwhich a nucleic acid sequence encoding the enzyme is linked to thevarious other vector sequences.

HRP has been expressed in target cells by transfecting a construct(pRK34-HRP) in which HRP c-DNA is fused to the signal sequence for thehuman growth hormone and the KDEL retention motif (Connolly CN, FutterCE, Gibson A, et al., Transport into and out of the Golgi complexstudied by transfecting cells with cDNAs encoding horseradishperoxidase. J. Cell Biol. 1994; 127: 641-652). The resulting HRP hasbeen shown to activate indole-3-acetic acid and produce toxic compounds(Greco, O. et al. Development of an enzyme/prodrug combination for genetherapy of cancer, Proc. Amer. Assoc. Cancer Res., 40, 478 (1999)).

ADEPT

For applications in ADEPT systems, an antibody directed against a tumourspecific marker is linked to the relevant enzyme, which may be modifiedas described above. The antibody may be monoclonal or polyclonal. Forthe purposes of the present invention, the term “antibody”, unlessspecified to the contrary, includes fragments of whole antibodies whichretain their binding activity for a tumour target antigen. Suchfragments include Fv, F(ab′) and F(ab′)₂ fragments, as well as singlechain antibodies. Furthermore, the antibodies and fragments thereof maybe humanised antibodies, e.g. as described in EP-A-239400.

The antibodies may be produced by conventional hybridoma techniques or,in the case of modified antibodies or fragments, by recombinant DNAtechnology, e.g. by the expression in a suitable host vector of a DNAconstruct encoding the modified antibody or fragment operably linked toa promoter. Suitable host cells include bacterial (e.g. E. coli), yeast,insect and mammalian cells. When the antibody is produced by suchrecombinant techniques the enzyme may be produced by linking a nucleicacid sequence encoding the enzyme (optionally modified as describedabove) to the 3′ or 5′ end of the sequence of the construct encoding theantibody or fragment thereof.

PDEPT

In order for the PDEPT approach to be followed, the chosen peroxidasemust be bound to a polymer which is water soluble, biocompatible,non-immunogenic and which does not eliminate the activity of the enzyme.PDEPT is likely to present less severe immunogenic effects than ADEPTdue to the masking of the foreign enzyme by the polymer. HRP has beensuccessfully bound to polyethylene glycol (PEG), as well as otherpolymers (Fortier G and Laliberté M, “Surface modification ofhorseradish peroxidase with polyethylene glycol(s) of various molecularmasses” Biotechnol Appl Biochem. 17:115-130, 1993; Laliberté M, et al,Surface modification of horseradish peroxidase with polyethyleneglycol(s) of various molecular masses (Mr): relationship between the Mrof polyethylene glycol and the stability of horseradish peroxidase-poly(ethylene glycol) adducts under various denaturing conditions.Biotechnol Appl. Biochem 20:394-413, 1994

PDT

The activation process in PDT can be highly site specific.

The direction and width of a laser beam can be controlled with greatprecision. Therefore, it can act upon a very limited area, minimisingdamage to neighbouring tissue.

Highly reactive and thus cytotoxic species can also result fromrelatively low energy activations. For example, a reactive excited stateof molecular oxygen, the singlet state, differs in only 90 kJ/mol fromits ground triplet state. However, this enables sufficientconcentrations of the toxic species to be formed by those sensitiserswhich absorb at wavelengths longer than 600 nm, (Carruth, J. A. S.,Clinical applications for photodynamic therapy, J Photochem Photobiol(1991) 9, 396-397).

The main limitation of this approach arises from the physics of lightitself and its interaction with human tissue. The ability of light topenetrate tissue has been found to be wavelength-dependent. Penetratingability increases with increasing wavelength but limitations arise dueto light scattering and reflection. In biological tissues the scatteringcoefficient, for example of red light, is much greater than theabsorption coefficient, (Carruth, J. A. S., Clinical applications forphotodynamic therapy, J Photochem Photobiol (1991) 9, 396-397; Kennedy,J. C. And Pottier, R. H, Endogenous protoporphyrin IX, a clinical usefulphotosensitizer for photodynamic therapy, J Photochem Photobiol (1992)14, 275-292). As a result, photons entering the tissue are scatteredseveral times before they are either absorbed or diffused. Although thismight be expected to increase the energy delivered to certain areas,internal reflection results in an exponential decrease of energy fluxwith increasing distance from the tissue-air interface. Theselimitations have been partially overcome in the treatment of relativelybulky tumours or when deeper penetration is necessary by the use ofmultiple interstitial optical fibres.

Several tumour types have been identified as potential targets for PDT.They include head and neck tumours, carcinomas of the bronchus,malignant brain tumours, superficial tumours of the bladder and vasculardisease, which have all shown promising responses in the clinic (Regula,J., Mac Roberts, A. J., Gorchein, A., Buonaccorsi, Thorpe, S. M.,Spencer, G. M., Hartfield, A. R. W. and Bown, S. G., Photosensitisationand photodynamic therapy of oesophageal, duodenal and colorectal tumoursusing 5-aminoleavulic acid induced protoporphyrin IX-a pilot study, Gut(1995) 36, 67-75).

Apoptosis induction in human tumour cells by photoproducts ofindole-3-acetic acid sensitized by riboflavin has recently beendescribed (Edwards, A. M., et al. Apoptosis induction in nonirradiatedhuman HL-60 and murine NSO/2 tumor cells by photoproducts ofindole-3-acetic acid and riboflavin. Photochemisty and Photobiology, 70,645-649, 1999).

The use of compounds of formula (I) enhances PDT. In PDT, photolysis ofphotosensitizers P, equation (1), results in the reaction of thegenerated triplet excited state ³P* with ground state oxygen, producingtoxic singlet oxygen, equation (2). One drawback with conventional PDTis that no toxicity occurs in anoxia. Without wishing to be bound bytheory, a compound of formula (I), illustrated as IAA below, is oxidisedby ³P* to generate an indole radical cation, equation (3) which, asdiscussed above, leads to toxic products, with or without theinvolvement of oxygen.P+hv→³P*  (1)³P*+O₂→P+¹O₂  (2)³P*+IAA→P+IAA.+  (3)The combination, either in aerobic or anoxic conditions, of a compoundof formula (I) with a photosensitizer should result in a lowerconcentration of photosensitizer being needed to achieve the sametoxicity. This would reduce the normal tissue damage from light exposureafter treatment, which can occur up to several weeks after treatment.The sensitizing effects of IAA derivatives should last only for a fewhours, until the IAA is excreted.

The technique of PDT as discussed above can be used by employing acombination of appropriate compounds of formula (I) andphotosensitizers. The preferred wavelength of light used is 500 to 800nm.

Applications of the Invention

Compounds of the invention can be used in vitro or in vivo for a rangeof applications. For example, for GDEPT a number of vector systems forthe expression of peroxidase in a cell have been developed. The furtherdevelopment of such systems (e.g. the development of promoters suitablefor specific cell types) requires suitable candidate prodrugs capable ofkilling cells when activated by peroxidase. Prodrug compounds of formula(I) susceptible to peroxidase may be used in such model systems. Themodel systems may be in vitro model systems or in vivo xenograft modelsystems comprising for example human tumour cells implanted in nudemice.

Compounds of formula (I) in conjunction with photosensitizers activatedby light having a wavelength between 500 and 800 nm may be tested invitro with other suitable forms of activation against panels ofdifferent tumour cell types to determine efficacy against such tumourcells. The efficacy of compounds of the invention against a range oftumour cell types may be used as points of reference for the developmentof further antitumour compounds.

Compounds of formula (I) may also be used in a method of treatment ofthe human or animal body. Such treatment includes a method of treatingthe growth of neoplastic cells in a patient with neoplastic diseasewhich comprises administering to a patient in need of treatmentcompounds of formula (I) as part of an ADEPT, GDEPT, PDEPT or PDT systemwhere neoplastic diseases include leukaemia and solid tumours such asovarian, colonic, lung, renal, breast, bowel, head and neck, CNS andmelanomas.

It will be understood that where treatment of tumours is concerned,treatment includes any measure taken by the physician to alleviate theeffect of the tumour on a patient. Thus, although complete remission ofthe tumour is a desirable goal, effective treatment will also includeany measures capable of achieving partial remission of the tumour aswell as a slowing down in the rate of growth of a tumour including itsmetastases. Such measures can be effective in prolonging and/orenhancing the quality of life and relieving the symptoms of the disease.

Therapies

Methods of ADEPT, GDEPT and PDEPT will now be described. The basis ofPDT has been described above, but the information on the administrationof products below also applies to this type of therapy.

ADEPT Therapy

The antibody/enzyme conjugate for ADEPT can be administeredsimultaneously with the prodrug but it is often found preferable, inclinical practice, to administer the enzyme/antibody conjugate beforethe prodrug, e.g. up to 72 hours or even 1 week before, in order to givethe enzyme/antibody conjugate an opportunity to localise in the regionof the tumour target. By operating in this way, when the prodrug isadministered, conversion of the prodrug to the cytotoxic agent tends tobe confined to the regions where the enzyme/agent conjugate islocalised, i.e. the region of the target tumour. In this way, thepremature release of the compound produced by the action of theperoxidase on the prodrugs of the present invention is minimised.

In ADEPT the degree of localisation of the enzyme/agent conjugate (interms of the ratio of localized to freely circulating active conjugate)can be further enhanced using the clearance and/or inactivation systemsdescribed in WO89/10140. This involves, usually following administrationof the conjugate and before administration of the prodrug, theadministration of a component (a “second component”) which is able tobind to part of the conjugate so as to inactivate the enzyme in theblood and/or accelerate the clearance of the conjugate from the blood.Such a component may include an antibody to the enzyme component of thesystem which is capable of inactivating the enzyme.

The second component may be linked to a macromolecule such as dextran, aliposome, albumin, macroglobulin or a blood group O erythrocyte so thatthe second component is restrained from leaving the vascularcompartment. In addition, or as an alternative, the second component mayinclude a sufficient number of covalently bound galactose residues, orresidues of other sugars such as lactose or mannose, so that it can bindthe conjugate in plasma but be removed together with the conjugate fromplasma by receptors for galactose or other sugars in the liver. Thesecond component should be designed for use and administered such thatit will not, to any appreciable extent, enter the extravascular space ofthe tumour where it could inactivate localised conjugate prior to andduring administration of the prodrug.

In ADEPT systems, the dose of the prodrug and conjugate will ultimatelybe at the discretion of the physician, who will take into account suchfactors as the age, weight and condition of the patient. Suitable dosesof prodrug and conjugate are given in Bagshawe et al., Antibody,Immunoconjugates, and Radiopharmaceuticals (1991), 4, 915-922. Asuitable dose of conjugate may be from 500 to 200,000 enzyme units/m²(e.g. 20,000 enzyme units/m²) and a suitable dose of prodrug may be fromabout 0.1 to 200 mg/Kg, preferably from about 10 to about 100 mg/Kg perpatient per day.

In order to secure maximum concentration of the conjugate at the site ofdesired treatment, it is normally desirable to space apartadministration of the two components by at least 4 hours. The exactregime will be influenced by various factors including the nature of thetumour to be targeted and the nature of the prodrug, but usually therewill be an adequate concentration of the conjugate at the site ofdesired treatment within 48 hours.

The targeting strategy may be enhanced in some combinations of drug andperoxidase by targeted delivery of the hydrogen peroxide co-factor tothe tumour. Targeted delivery of either polymer- or antibody-boundglucose oxidase is described by, e.g., Samoszuk M, Emerson J, Nguyen V,et al. “Targeting of glucose oxidase to murine lymphoma allografts”.,Tumor Targeting (1995) 1, 37-43; Ben-Yoseph O, Ross BD., “Oxidationtherapy: the use of a reactive oxygen species-generating enzyme systemfor tumour treatment.”, Br J Cancer (1994) 70, 1131-1135.

The antibody/enzyme conjugate may be administered by any suitable routeusually used in ADEPT therapy. This includes parenteral administrationof the antibody in a manner and in formulations similar to thatdescribed below.

PDEPT Therapy

As in ADEPT therapy, the polymer/enzyme conjugate can be administeredsimultaneously but it is often found preferable, in clinical practice,to administer the polymer/enzyme conjugate before the prodrug, e.g. upto 72 hours or even 1 week before, in order to give the polymer/enzymeconjugate an opportunity to localise in the region of the tumour target,and hence avoid reaction of the enzyme and prodrug not at the desiredsite of action.

The dosing and administration of the polymer/enzyme and prodrug will beas described above for ADEPT.

GDEPT Therapy

For use of the vectors in therapy, the vectors will usually be packagedinto viral particles and the particles delivered to the site of thetumour, as described in for example Ram et al. (ibid). The viralparticles may be modified to include an antibody, fragment thereof(including a single chain) or tumour-directed ligand to enhancetargeting of the tumour. Alternatively the vectors may be packaged intoliposomes. The liposomes may be targeted to a particular tumour. Thiscan be achieved by attaching a tumour-directed antibody to the liposome.Viral particles may also be incorporated into liposomes. The particlesmay be delivered to the tumour by any suitable means at the disposal ofthe physician. Preferably, the viral particles will be capable ofselectively infecting the tumour cells. By “selectively infecting” it ismeant that the viral particles will primarily infect tumour cells andthat the proportion of non-tumour cells infected is such that the damageto non-tumour cells by administration of a prodrug will be acceptablylow, given the nature of the disease being treated. Ultimately, thiswill be determined by the physician.

One suitable route of administration is by injection of the particles ina sterile solution. Viruses, for example isolated from packaging celllines, may also be administered by regional perfusion or directintratumoral injection, or direct injection into a body cavity(intracaviterial administration), for example by intra-peritonealinjection.

The exact dosage regime for GDEPT will, of course, need to be determinedby individual clinicians for individual patients and this, in turn, willbe controlled by the exact nature of the prodrug and the cytotoxic agentto be released from the prodrug. However, some general guidance can begiven. Chemotherapy of this type will normally involve parenteraladministration of modified virus, and administration by the intravenousroute is frequently found to be the most practical. The GDEPT approachmay be combined with radiotherapy to further enhance efficacy. WhenGDEPT is combined with radiotherapy, the combination of the compound offormula (I) with the GDEPT system may be considered a radiosensitizer.

In GDEPT systems the amount of virus or other vector delivered will besuch as to provide a high enough cellular concentration of enzyme so asto catalyse the efficient conversion of prodrugs to cytotoxins.Typically, the vector will be administered to the patient and then theuptake of the vector by transfected or infected (in the case of viralvectors) cells monitored, for example by recovery and analysis of abiopsy sample of targeted tissue. This may be determined by clinicaltrials which involve administering a range of trial doses to a patientand measuring the degree of infection or transfection of a target cellor tumour. The amount of prodrug required will be similar to or greaterthan that for ADEPT systems.

In using a GDEPT system the prodrug will usually be administeredfollowing administration of the vector encoding an enzyme. Suitabledoses of prodrug are from about 0.1 to 200 mg/Kg, preferably from about10 to about 100 mg/Kg per patient per day.

Administration of Prodrugs

While it is possible for the compounds of formula (I) to be administeredalone, it is preferable to present them as pharmaceutical formulations,for use with any of the above methods. The formulations comprise thecompounds, together with one or more acceptable carriers thereof andoptionally other therapeutic ingredients, or diluents. The carrier orcarriers must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipients thereof, for example, liposomes. Suitable liposomes include,for example, those comprising the positively charged lipid(N[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA), thosecomprising dioleoylphosphatidylethanolamine (DOPE), and those comprising3β[N-(n′N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol).

Formulations suitable for parenteral or intramuscular administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats, bacteriocidal Antibioticsand solutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents, and liposomesor other microparticulate systems which are designed to target thecompound to blood components or one or more organs. The formulations maybe presented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of a sterile liquid carrier, forexample Water for Injection, immediately prior to use. Injectionsolutions and suspensions may be prepared extemporaneously from sterilepowders, granules and tablets of the kind previously described.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations may include other agents conventionalin the art having regard to the type of formulation in question. Of thepossible formulations, sterile pyrogen-free aqueous and non-aqueoussolutions are preferred.

The doses may be administered sequentially, e.g. at hourly, daily,weekly or monthly intervals, or in response to a specific need of apatient. Preferred routes of administration are oral delivery andinjection, typically parenteral or intramuscular injection orintratumoural injection. For methods of PDT dermal or topicaladministration may be preferred, e.g. subcutaneous injection or creamsand ointments, and such methods of administration are well known.

The exact dosage regime will, of course, need to be determined byindividual clinicians for individual patients and this, in turn, will becontrolled by the exact nature of the compound of formula (I), but somegeneral guidance can be given. Typical dosage ranges generally will bethose described above which may be administered in single or multipledoses. Other doses may be used according to the condition of the patientand other factors at the discretion of the physician.

The following examples illustrate the invention.

Experimental Details

All air-sensitive reactions were carried out in a nitrogen atmosphere.Glassware was oven-dried and cooled in an anhydrous atmosphere prior touse. The melting points were determined using a Gallenkamp melting pointapparatus, and are uncorrected. NMR spectra (60 MHz) were recorded on aJeol MY60 spectrometer. Mass spectra were recorded on a Waters IntegrityHPLC/MS system.

EXAMPLE 1

General Method for Fischer Indole Synthesis (Engvild KC et al., ibid.)(See Scheme 1 for Outline)

L-Glutamic acid (30 mmol) (or the appropriate derivative)was dissolvedin an equimolar quantity of 2M sodium hydroxide and cooled to 0° C.Sodium hypochlorite solution (1 equivalent) was added, and the mixturestirred at 0° C. for 1 hour. 3M hydrochloric acid (3 equivalents) wasthen added, and the mixture stirred at 55° C. until negative tostarch-iodide. The substituted phenylhydrazine hydrochloride (typically10 mmol) was added to the warm solution, and the mixture stirred for 1hour whilst cooling to room temperature. The mixture was then extractedwith ethyl acetate (3×50 ml), the combined organic extracts dried overmagnesium sulfate, and the solvent removed in vacuo to furnish the crudephenylhydrazone.

This product was then dissolved in pyridine (24 ml). Conc. hydrochloricacid (31 ml) was added, followed by 85% phosphoric acid (8-10 ml), andthe mixture heated under reflux in an inert atmosphere for 18 hours. Thecooled reaction mixture was poured into an equal volume of ice-coldwater, and then extracted with diethyl ether (3×75 ml), the combinedether layers dried (MgSO₄) and the solvent removed in vacuo to give thecrude product, which was purified by recrystallisation from chloroform.

Example 1(a) 5-Chloroindole-3-acetic acid (SR024)

Off-white needles, yield 11%, m.p. 160-161° C. (lit. 158-159° C.); m/z209 (M⁺), 164 (M⁺-CO₂H), 128; δH/ppm (CDCl ₃) 7.66 (1H, s), 7.19 (3H,m), 3.74 (2H, s); found C 57.05%, H 3.82%, N 6.62% (calculated C 57.30%,H 3.85%, N 6.68%).

Example 1(b) 7-Fluoroindole-3-acetic acid (SR022)

Beige powder, yield 24%, m.p. 159-161° C. (lit. 161-162° C.) m/z 193(M⁺), 148 (M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.63-6.78 (4H, m, ArH), 3.71 (2H,s, ArCH₂).

Example 1(c) 5-Trifluoromethoxyindole-3-acetic acid (SR073)

White powder, yield 7%, m.p. 120-122° C.; m/z 259 (M⁺), 214 M⁺-Co₂H);δH/ppm (CDCl₃) 7.43-7.11 (4H, m, ArH), 3.76 (2H, s, ArCH₂); found C50.96%, H 3.18%, N 5.40% (calculated C 50.98%, H 3.11%, N 5.40%).

Example 1(d) 5-Fluoro-2-methylindole-3-acetic acid (SR036)

White plates, yield 10%, m.p. 182-184° C. (lit. 179-182° C.); m/z 207(M⁺), 162 (M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.24-6.60 (3H, m, ArH), 3.65 (2H,s, ArCH₂), 2.36 (3H, s, 2-Me).

Example 1(e) 7-Fluoro-5-methylindole-3-acetic acid (SR109)

Off-white powder, yield 5%, m.p. 138-140° C.; m/z 207 (M⁺), 162(M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.29-7.13 (2H, m, ArH), 6.78-6.56 (1H, m,ArH), 3.69 (2H, s, ArCH₂), 2.38 (3H, s, 5-Me); found C 62.89%, H 4.86%,N 6.57% (calculated C 63.76%, H 4.86%, N 6.76%).

Example 1(f) 7-Fluoro-4-methylindole-3-acetic acid (SR148)

Pale beige powder, yield 4%, m.p. 162-164° C. dec., m/z: 207 (M⁺), 162(M⁺-CO₂H); 6H/ppm (CD₃COCD₃) 7.25 (1H, m, ArH), 6.71 (1H, m, ArH), 6.56(1H, ArH), 3.87 (2H, s, ArCH₂), 2.58 (3H, s, 4-CH₃),

Example 1(g) 4,6-Dichloroindole-3-acetic acid (SR085)

Off-white needles, yield 5%, m.p. 223-224-C (lit. 210-214° C.); m/z 245,243 (M⁺), 200, 198 (M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.31 (2H, br, ArH [H2,H7]), 6.98 (1H, s, ArH [H5]), 3.97 (2H, s, ArCH₂).

Example 1(h) 5-Chloro-7-fluoroindole-3-acetic acid (SR091)

White powder, yield 4%, m.p. 164-166° C.; m/z: 229, 227 (M⁺), 184, 182(M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.69-6.76 (3H, m, ArH), 3.72 (2H, s,ArCH₂); found C 51.57%, H 3.26%, N 6.01%, (calculated C52.77%, B 3.10%,N 6.15%, calculated C₁₀H₇ClFNO₂.0.25H₂° C. 51.72%, B 3.26%, N 6.04%).

Example 2

General Method for the Synthesis of Substituted Indole-Acetic Acids Fromthe Parent Substituted Indole

(Dillard, RD, et al., ibid.) (see scheme 2)

The substituted indole, which may be commercially available orsynthesised—see below, (typically 5-10 mmol) was dissolved in dry THF(20 ml) and cooled to 0° C. n-Butyllithium (1.1 equivalents) was added,and the mixture stirred at 0° C. for 20 minutes. Zinc chloride (1.1 eqof a 1M solution in diethyl ether) was added, and the mixture stirred atroom temperature for 2 hours. Ethyl bromoacetate (1.1 equivalents) wasthen added over 5 minutes, and the mixture stirred for a further 24hours. Water (20 ml) was added, and the mixture then extracted withethyl acetate (3×30 ml), the combined organic extracts dried (MgSO₄) andthe solution concentrated in vacuo to give the crude product. The crudematerial was purified by flash column chromatography (3:1 hexane:EtOAc)to give the substituted ethyl indolyl-3-acetate in moderate to lowyield, with significant recovery of starting material in most cases.Saponification was effected by heating the ester under reflux for 4hours in 10% methanolic NaOH (40 ml), then acidifying the mixture with1M hydrochloric acid, and filtering off the precipitated indole-3-aceticacid. Further purification was achieved by recrystallisation fromchloroform.

Example 2(a) 7-Chloroindole-3-acetic acid (SR052)

From commercially available starting material) Off-white powder, yield11%, m.p.164-165-C (lit. 163-165° C.); m/z: 211, 209 (M⁺), 164(M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.61-6.99 (4H, m, ArH), 3.77 (2H, s,ArCH₂); found C 55.51%, H 3.77%, N 6.42% (calculated C 57.30%, H 3.85%,N 6.68%).

Example 3

General Method for Leimgrüber-Batcho Synthesis of the Indole

(Batcho, AD, et al., ibid.) (see scheme 3 for outline)

5 g of the halonitrotoluene was heated with N,N-dimethylformamidedimethyl acetal (1.1 equivalents) and pyrrolidine (1.1 equivalents)until the reaction was judged to be complete by thin layerchromatography. Addition of methanol and concentration under reducedpressure gave the β-pyrrolidinostyrene as either a dark red oil or acrystalline solid, which was then subjected to reduction without furtherpurification.

The β-pyrrolidinostyrene (ca 20 mmol) was dissolved in methanol (50 ml).Raney nickel (0.5 ml of 50% w/v in water) and hydrazine hydrate (30mmol) were added. The mixture was stirred at 45° C. for 30 minutes, andthen a further portion of hydrazine hydrate (30 mmol) was added, and themixture stirred at 45-50° C. for a further 1 hour. The reaction mixturewas filtered through celite and the filtrate evaporated to give a brownoil, which was purified by flash column chromatography (4:1hexane:EtOAc) to furnish the resulting indole. The indole-3-acetic acidwas then synthesised via the ethyl ester, by the method described inexample 2 above.

Example 3(a) 4-Fluoroindole-3-acetic acid (SR039)

Brownish-white prisms, yield 22%, m.p. 129-131° C. (lit. 128-129° C.);m/z: 193 (M⁺), 148 (M⁺-CO₂H); δH/ppm (CD₃COCD₃): 7.23-6.49 (4H, m, ArH),3.85 (2H, s, ArCH₂); found C: 61.87%, H: 4.19%, N: 7.12% (calculated C:62.18%, H: 4.17%, N: 7.25%).

Example 3(b) 6-Fluoroindole-3-acetic acid (SR043)

Off-white needles, yield 30%, m.p. 161-163° C. (lit. 165° C.); m/z 193(M⁺), 148 (M⁺-CO₂H), 101; δH/ppm (CD₃COCD₃): 7.61-6.43 (4H, m, ArH),3.73 (2H, s, ArCH₂); found C: 62.08%, H: 4.18%, N: 7.22% (calculated C:62.18%, H: 4.17%, N: 7.25%).

Example 3(c) 6-Chloroindole-3-acetic acid (SR058)

Pale pink plates, yield 14%, m.p. 183-185° C. (lit. 182-184° C.); m/z209 (M⁺), 164 (M³⁰-CO₂H); δH/ppm 7.66-7.06 (4H, m, ArH), 3.71 (2H, s,ArCH₂); found C 57.38%, H 3.84%, N 6.67% (calculated C 57.30%, H 3.85%,N 6.68%).

Example 3(d) 6-Bromoindole-3-acetic acid (SR069)

Pale pink lustrous flakes, yield 12%, m.p. 174-176° C. (lit. 172-177°C.); m/z 255,253 (M⁺), 210,208 (M⁺-CO₂H); δH/ppm (CD₃COCD₃)7.61-7.12(4H, m, ArH), 3.76 (2H, s, ArCH₂); found C 44.79%, H 3.20%, N 5.11%(calculated C 47.27%, H 3.17%, N 5.51%).

Example 4

General Method for Preparation of Arylindoles by Suzuki Coupling ofBromoindoles

(see scheme 5 for outline)

The appropriate bromoindole (2-5 mmol) andtetrakis(triphenylphosphine)-palladium (0) (5 mol %) were stirred intoluene (15 ml) under nitrogen for 5 minutes, and then the arylboronicacid (1.1 equivalents) in ethanol (4 ml) was added, followed by aqueoussodium carbonate (2 equivalents), and the mixture was then heated at 80°C. under nitrogen for 24 hours. Water (20 ml) was added, and the mixtureextracted with ethyl acetate (3×50 ml), the organic layers dried (MgSO₄)and the solvent removed in vacuo to give the crude product, which waspurified by column chromatography (3:1 hexane:EtOAc) to give the purearylindole. The indole-3-acetic acid was then synthesised via the ethylester, as described in example 2 above.

Example 4(a) 5-(4-Chlorophenyl)indole-3-acetic acid (SR150)

White flakes, yield 15%, m.p. 166-168° C.; m/z 287, 285 (M⁺), 242, 240(M⁺-CO₂H); δH/ppm (CD₃COCD₃) 7.84-7.28 (8H, m, ArH), 3.77 (2H, s,ArCH₂); found C 67.05%, H 4.20%, N 4.83% (calculated C 67.26%, H 4.23%,N 4.90%).

Example 5

General Method for Synthesis of Indoles by Bartoli Method

(Bartoli G, et al., ibid.) (see scheme 4 for outline)

The appropriate 2-substituted nitrobenzene (typically 5-10 mmol) wasdissolved in THF (20 ml) and cooled to −40° C. Vinylmagnesium bromide (3equivalents of a 1 M solution in ether) was added, and the mixturestirred at −40° C. for 20 minutes, then poured into saturated ammoniumchloride solution. The mixture was extracted with diethyl ether (3×50ml) and the combined organic layers dried (MgSO₄) and the solventremoved in vacuo to give the crude indole, which was purified by flashcolumn chromatography. The resulting indole intermediates can beconverted to the corresponding indole-3-acetic acids by the methoddescribed in example 2.

Example 5(a) 7-Bromoindole-3-acetic acid

7-bromoindole: Pale brown needles, yield 24%, m.p. 42-44° C. (lit(Aldrich chemical catalogue) 41-44° C.); m/z 197, 195 (M⁺).

Example 5(b) 6,7-Dichloroindole-3-acetic acid

6,7-dicholorindole: Off-white fluffy needles, yield 25%, m.p. 50-52° C.;m/z 185, 187, 189 (ratio 9:6:1, M⁺), 150, 152 (M⁺-Cl).

Example 6

N-Substituted INDOLE-3-acetic acids

Example 6(a) 6-Chloro-1-methylindole-3-acetic acid (SR139)

Sodium hydride (0.19 g of 60% suspension in oil) was suspended in dryTHF (10 ml). 6-Chloroindole-3-acetic acid (SR058)(100 mg, 0.48 mmol) inTHF (5 ml) was added dropwise and the mixture then stirred for 10minutes. Iodomethane (0.34 g, 2.4 mmol) was then added, and the mixturestirred overnight. Excess sodium hydride was carefully destroyed bydropwise addition of water, and then the mixture was acidified (1M HCl)and extracted with ethyl acetate (3×50 ml), the organic layers driedover MgSO₄, and the solvent evaporated to give a brown powder, yield 75mg, 70%, m.p. 130-132° C. dec.; m/z 225, 223 (M⁺), 180, 178 (M⁺-CO₂H);δH/ppm 7.46-6.92 (4H, m, Ar—H), 3.77 (3H, s, N—Me), 3.70 (2H, s, ArCH₂).

Example 6(b) 5-Fluoro-1-methylindole-3-acetic acid (SR164)

Synthesised as in example 6(a). White needles, yield 52%; m.p 134-136°C.; m/z 207 (M⁺), 162 (M⁺-CO₂H).

Example 7 Measurements of the Cytotoxic Effects of Treatment withCompounds of Formula (I) and Horseradish Peroxidase (HRP) Using Hamster(V79) and Human (MCF7 and HT29) Cell Lines in vitro Example 7(a) V79Cells

Hamster lung fibroblast V79 cells were obtained from the European TissueCulture Collection. Cells were grown over the weekend in 75 mL tissueculture flats as attached cells in Eagle's Modified Essential Medium(EMEM) supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100U/mL penicillin and 100 μg/mL streptomycin, and incubated in ahumidified incubator at 37° C., in 5% CO₂/air. Cells were removed bytrypsin treatment when a confluent layer had grown: the cells werewashed with phosphate buffered saline and 2 mL of 0.5% w/v porcinetrypsin with 0.2% w/v EDTA added for a few minutes at room temperatureuntil the cells were loosened. The trypsin was deactivated by theaddition of 10 mL spinner modified EMEM (supplemented with 7.5% foetalcalf serum) and the cells allowed to grow in a spinning culture,pre-gassed with 5% CO₂/air, maintaining the cells in logarithmic growth.

Cells were prepared for toxicity measurements by counting and plating200, 2000 or 20,000 cells/6 cm Petri dish in 3 mL EMEM in triplicate foreach sample. The cells were allowed to attach for at least 1 hour, thenthe medium removed and 2 mL phenol red free Hanks' balanced saltsolution added, either alone (control) or with 50 or 100 μM of thecompound of formula (I) to be tested. HRP (50 μL of 0.048 mg/mL) wasadded to the test dishes requiring it and the cells left in theincubator for 0, 0.5, 1, 1.5 or 2 hours. After the incubation time thedrug was removed, the cells washed with 2 mL Hanks and then 4 mL EMEMadded. The cells were allowed to grow for 7 days to colonies of >50cells. After 7 days the medium was removed and the cells fixed with 75%methanol for 5 minutes. The methanol was then removed and the cellsstained with 1% w/v crystal violet for 5 minutes. The cells were washedand colonies counted. Cell survival was determined by calculating theratio of colonies growing compared to untreated control cells.

In table 1, values are the surviving fraction after 2 hours treatmentwith the IAA derivatives at the concentrations shown together with HRP(1.2 μg/mL). Unless otherwise indicated (by n=number of individualexperiments), the means±the standard errors of three experiments aregiven. No detectable cytotoxic effects were seen at these times andconcentrations with IAA derivative alone or with HRP alone.

TABLE 1 Compound Substituent(s) 50 μM 100 μM none*  0.013 ± 0.00481-methyl* 0.013 ± 0.005 2-methyl* 0.0042 ± 0.0024 2-methyl-5- 0.044 ±0.013 methoxy* 5-methoxy* 1^(b) 5-bromo^(c) 0.00004 ± 0.00003 SR0696-bromo 0.0031 ± 0.0023 0^(a) SR024 5-chloro 0.0027 ± 0.0022 SR0586-chloro 0.00085 (n = 1) 0^(a) SR052 7-chloro 0.000042 (n = 1) SR0394-fluoro 0.000085 ± 0.00007  5-fluoro^(c) 0.0045 ± 0.003  0^(a) SR0436-fluoro 0.00995 (n = 2) SR022 7-fluoro 0.053 ± 0.022 0^(a) (90% pure)*= comparative example ^(a)= all cells killed ^(b)= no detectable effect^(c)= commercially availableFIGS. 1 and 2 relate to experiments carried out using5-fluoro-indole-3-acetic acid. FIG. 1 shows the variation in thesurviving fraction of cells against varying concentration of5-fluoro-indole-3-acetic acid, following 5 hours incubation, with orwithout HRP (1.2 μg/L). FIG. 2 shows the variation in the survivingfraction of cells against the varying concentration of HRP, following 5hours incubation, with 50 μM of 5-fluoro-indole-3-acetic acid.

Example 7(b) Human Cell Lines

Human breast carcinoma cells (MCF7) and human colon carcinoma cells(HT29) were obtained from the European Tissue Culture Collection. Cellswere grown as attached monolayers in EMEM supplemented with 10% foetalcalf serum, 2 mM L-glutamine, 5% w/v non-essential amino acids, 100 U/mLpenicillin and 100 μg/mL streptomycin, and incubated in a humidifiedincubator at 37° C., in 5% CO₂/air. Cell survival experiments werecarried out exactly as for V79 cells except that cells were plated intoPetri dishes directly from a trypsinised confluent flat and allowed toplate for at least 4 hours before addition of the drug.

FIG. 3 shows the variation in the surviving fraction of MCF7 and HT29cells when incubated for varying lengths of time with 100 μM5-fluoro-indole-3-acetic acid and with or without HRP (1.2 μg/L).

Example 8 Measurements of the Cytotoxic Effects of Treatment withCompounds of Formula (I) and Horseradish Peroxidase (HRP) Bound to aPolymer (Polymer Directed Enzyme-Prodrug Therapy, PDEPT)

Preparation of the HRP-Polymer Conjugate

HRP was conjugated to polyethylene glycol (PEG) based on the method ofFortier and Laliberté (1993, ibid.). HRP (7.82 mg) was mixed withmethoxypoly(ethylene glycol)-nitrophenyl carbonate (PEG, ShearwaterPolymers, Huntsville, Ala., USA) molecular weight 5240 Da (42.66 mg) in4 mL 0.1 M borate buffer pH 9.4 overnight at 4° C. The following day 50mL 35 mM sodium phosphate buffer pH 6.5 was added and the resultingyellow p-nitrophenol removed by filtering through a Vectraspin (Whatman)20 kDa cut off filter. The resulting concentrated mixture of HRP andHRP-PEG conjugate was stored overnight in a refrigerator. The proteinswere separated on a Sephadex G 75 superfine column (2×35 cm) with 25 mMsodium acetate pH 5 at 1 mL/h, 4° C. and 1 hour fractions collected witha fraction collector. HRP was measured spectrophotometrically at 405 nm.The purity of the fractions was measured on a SDS-PAGE gel (8% agarosegel with 4% stacking gel) and the bands stained with Coomassie Blue(0.25 g Coomassie Blue in 10 mL glacial acetic acid and 90 mL 50%methanol) for 50 minutes. The gel was destained overnight with 10 mLglacial acetic acid and 90 mL 50% methanol. Approximately 10 mg pureHRP-PEG conjugate was obtained with a range of molecular weights up to25 kDa greater than pure HRP. The resulting protein was dried undernitrogen and stored under nitrogen at −20° C.

Measurement of the Peroxidase Activity of the HRP-Polymer. Conjugate

Activity of the HRP-PEG was measured using the Sigma ABTS(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) method. ABTS(0.725 mL of 9.1 mM in 0.1M potassium phosphate buffer pH 5), 12.5 μMHRP solution (approximately 20 μg/ml in 40 mM phosphate buffer with0.25% bovine serum albumin and 0.5% w/v Triton X-100 pH 6.8), and 25 μL0.3% hydrogen peroxide were mixed, and the rate of increase inabsorbance at 406 nm measured (with a 400 nm cut off filter) on aHewlett Packard 8452A diode array spectrophotometer. Assuming anextinction coefficient of 36,800 M⁻¹ s⁻¹, the activity of HRP/PEG wascalculated from the unit definition: 1 unit HRP will oxidise 1 μmol ofABTS per minute at pH 5, 25° C.

Biodistribution Studies of the HRP-Polymer Conjugate

Experiments to measure tissue uptake of HRP-PEG were carried out infemale CBA/Gy mice with a subcutaneous dorsum breast adenocarcinomatumour (CaNT). Tumours arose from inoculation with a 0.5 mL suspensionof a disaggregated solid tumour under an inhalation anaesthetic. Tumourswere used approximately 3 weeks later (the tumours had an average massof about 0.4 g).

Mice were injected intra-peritoneally (i.p.) with 0.3 mL 0.5 mg/mLHRP-PEG, intra-tumour (i.t.) with 50 μL 3 mg/mL HRP-PEG orintra-venously (i.v.) with 100 μL 1.5 mg/mL HRP-PEG. The mice werekilled by cervical dislocation up to 48 hours later, the tissuesremoved, weighed and stored at −20° C. Blood was removed from the chestcavity, spun down and the plasma stored at −20° C.

HRP-PEG tissue accumulation was measured using tetramethylbenzidine(TMB). Tissues were macerated in 2 mL 0.5% hexadecyltrimethyl ammoniumbromide in 50 mM phosphate buffer (pH 6) and freeze-thawed twice. Thesuspensions were heated at 60° C. for 2 h, centrifuged at 4500 rpm for 5mins, and stored at −20° C. Enzyme activity was measured by mixing 1.87mL 80 mM phosphate buffer pH 5.4, 70 μL 88 mM hydrogen peroxide, 20 μL 1mg/mL TMB in 10% dimethyl sulphoxide (DMSO) and 40 μl sample. The rateof increase in absorbance at 630 nm was measured with a Hewlett Packard8452A diode array spectrophotometer. The absorbance change per minutewas calculated relative to protein content of the samples which weremeasured by a Bio Rad Lowry method after being diluted 10-fold in water.Enzyme levels were measured relative to control levels which allowedinaccuracies from variations in haemoglobin content to be eliminated.Uptake of HRP-PEG into the tumour occurred after i.p. and i.t. injectionalthough liver uptake was high. Injection of HRP-PEG i.t. allowedactivity to be measured in the tumour up to 20 hours later. Withoutwishing to be bound by theory, liver uptake is thought to be due tomannose glycoprotein receptors on hepatocyte cells selectively taking upHRP (which is a mannose glycoprotein) from the circulation, which may beavoided by protein modification without affecting the activity (J WTams, K G Welinder, Anal. Biochem. 1995, 228, 48-55).

Example 9 Measurements of the Cytotoxic Effects of Treatment withCompounds of Formula (I) and Horseradish Peroxidase (HRP) Bound to anAntibody (Antibody Directed Enzyme-Prodrug Therapy, ADEPT)

The Tumour Xenograft Used

The human colon adenocarcinoma cell line LS174T was used to develop axenograft model in the flanks of MF1 nude mice. Subsequent passaging wasby subcutaneous implantation of small tumour pieces (approximately 1mm³). The tumour is a poor- to moderately-differentiatedcarcinoembryonic antigen-(CEA)-producing adenocarcinoma with smallglandular acini, which secretes no measurable CEA into the circulation.All mice used were females aged 2-3 months and weighing 20-25 g.

The Antibody Used

The A5B7 antibody was used. This is a monoclonal anti-CEA antibody,which gives positive staining for glandular luminal surface andcytoplasm in the LS174T xenograft model. The antibody was labelled with¹²⁵I using the iodogen method, to a specific activity of 37 MBq/1 mgprotein, and sterilised by passing through a 0.22 mm filter (Gelman,Northampton, UK).

Preparation of the Antibody-Enzyme Conjugate

The method of Nakane and Kawaoi (1974) as modified by Hudson and Hay(1980) was used. The A5B7 antibody was dialysed three times against 0.2M sodium carbonate buffer pH 9.5 at 4° C. HRP (Sigma Type VI, RZ˜3.0, 4mg) was dissolved in 1.0 ml deionized water. Freshly prepared sodiumperiodate (200 μL, 0.1 M) was added to the mixture and stirred gentlyfor 20 minutes at room temperature to activate the HRP. The solution wasdialysed overnight against sodium acetate (1 L, 1 mM pH 4.4) at 4° C.Carbonate buffer (20 μL, 0.2 M) was added to the dialysed HRP aldehyde,raising the pH to 9-9.5 just before addition of the antibody to belabelled. The solution was incubated at room temperature for 2 hourswith gentle stirring. Sodium borohydride (100 μL 4 mg/ml freshlyprepared) was added and left for 2 hours at 4° C. to reduce the freeenzyme. The solution was then dialysed overnight against borate buffer(2 L 0.1 M pH 7.4). The labelled antibodies were stored at 4° C.

Studies of the Biodistribution of the Antibody-Enzyme Conjugate

The conjugate labelled with ¹²⁵I was used in some experiments. Usinggroups of 4 mice, antibody and antibody-enzyme distribution wasinvestigated at 6, 24 and 48 hours after antibody injection. All micereceived 0.75 MBq/20 g ¹²⁵I-A5B7 i.v. into the tail vein. At theselected time points the mice were bled, and liver, kidney, lung,spleen, colon, muscle and tumour removed for comparative activityassessment. Each tissue was weighed, digesting in 7 M KOH, and countedby gamma counter (Wizard: Pharmacia, Milton Keynes, UK). Results wereexpressed as percentage injected dose per g tissue, and the groupscompared using the Mann-Witney U test. Mice were given food and water adlibitum, the water containing 0.1% potassium iodide to block thyroiduptake of iodine.

In other experiments the enzyme activity of the HRP-A5B7 conjugate wasmeasured. Using two groups of four LS174T tumour-bearing nude mice,antibody-enzyme activity and distribution was measured in tissues 24 and72 hours after antibody injection. All mice were injected i.v. with 250μg HRP-A5B7 into the tail vein. Mice were bled at the selected timepoints and the tumour, liver, lung, kidney, muscle and plasma removedand stored at −20° C. Enzyme activity was measured using the tetramethylbenzidine method described above for assay of the HRP-PEG conjugate.Activity was measured relative to untreated control tumour-bearing mice.

Example 10 Measurements of the Cytotoxic Effects of Treatment withCompounds of Formula (I) Combined with Photosensitizers and VisibleLight in Photodynamic Therapy (PDT)

V79 hamster cells were photolysed in the presence of a variety ofphotosensitizers and compounds of formula (I).

V79 cells were plated for at least 1 hours in EMEM on 6-well plates induplicate in phenol free DMEM. The medium was removed from the cells inthe dark and 2 mL 2 μM of the photosensitzer and 0.1 mM of the testcompound (5-fluoro IAA and 5-bromo IAA) added in the dark andimmediately photolysed for varying lengths of time using a matrix oflight-emitting diodes at the appropriate wavelength for thephotosensitizer. The cells were left in an incubator for 1 hour, thedrugs removed, the cells washed with 2 mL PBS solution in the dark andthen left to grow for 6 days in 3 mL EMEM. Colonies of >50 cells werecounted after fixing and staining with crystal violet (see above).

The results in terms of surviving fraction of cells (relative tountreated control cells) against increasing photolysis time are shown inthe figures indicated below.

Photo- Methylene Toluidine Rose sensitizer Blue Thionine Blue BengalWavelength of 660 630 630 574 irradiating light (nm) Figure for 4a 4b 4c4d resultsVery little toxicity was seen with the photosensitizer or IAA alone atthe same concentrations. Toxicity may be due to the formation of theputative cytotoxins, 3-methylene-2-oxindoles which were shown to beformed by high performance liquid chromatography.

Example 11 Measurements of the Cytotoxic Effects of Treatment withCompounds of Formula (I) and Horseradish Peroxidase (HRP) Using CellsTransiently Transfected with the HRP Encoding Gene (Gene DirectedEnzyme-Prodrug Therapy, GDEPT)

Human bladder carcinoma T24 cells, MCF-7 mammary carcinoma cells (bothfrom European Collection of Cell Cultures, Salisbury, UK), FaDu,nasopharyngeal squamous carcinoma cells (American Type CultureCollection, Manassas, Va.) were maintained in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% foetal calf serum, 2 mML-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and incubatedin a humidified incubator at 37° C. and 5% CO₂/air. Only cells whichtested negative for mycoplasma infection were utilised.

The plasmid pRK34-HRP containing the HRP cDNA was provided by Dr D FCutler, University College, London (Connolly CN, Futter CE, Gibson A, etal., Transport into and out of the Golgi complex studied by transfectingcells with cDNAs encoding horseradish peroxidase. J. Cell Biol. 1994;127: 641-652).

Transient transfectants were obtained by exposing the cells to complexesof integrin-targeted peptides, lipofectin and DNA (Hart SL,Arancibia-Cárcamo CV, Wolfert MA, et al. Lipid-mediated enhancement oftransfection by a nonviral integrin-targeting vector. Human Gene Ther.1998; 9: 575-585) and were assayed for gene expression after 24 h.Transfection efficiency was estimated at about 20% for T24 cells, 16-20%for MCF-7 cells and 10-14% for FaDu cells.

Exponential growing transfected and untransfected cells were counted andplated at low density on Petri dishes and exposed to compounds offormula (I) for 24 hours in phenol-red-free Hanks' balanced saltsolution (HBSS) in a 37° C. incubator.

Alternatively, to test activity in hypoxic conditions, T24 cells werepre-plated in an anaerobic cabinet and, after incubation for 5 to 6hours to ensure anoxic conditions, were exposed to compounds of formula(I) for 24 hours in the cabinet. All plastics and media werepre-incubated in anoxia for 48 hours before use to remove residualoxygen.

Following drug exposure, cells were rinsed with PBS and grown for 8 to20 days in complete DMEM supplemented with feeder cells (V79 cellsexposed to 250 Gy ⁶⁰Co irradiation). After fixation and staining with2.5% crystal violet w/v in methylated spirit, colonies of >50 cells werescored. Surviving fractions were evaluated relative to HBSS-treatedcontrols.

FIG. 5 shows the variation in the surviving fraction of transfected andcontrol T24 cells in oxic conditions against varying concentrations of5-fluoro-indole-3-acetic acid or 5-bromo-indole-3-acetic acid.

A concentration of test compound to reduce cell survival by 50% (IC₅₀)was estimated from the survival curves, and are tabulated below, whereHRP⁺ represents transfected cells, HRP⁻ untransfected cells, and factorthe ratio of IC₅₀ for transfected cells to untransfected cells.

T24 (normoxic) MCF-7 FaDu T24 (anoxic) HRP⁺ HRP⁻ factor HRP⁺ HRP⁻ factorHRP⁺ HRP⁻ Factor HRP⁺ HRP⁻ factor 5F 0.04  3.5  88 0.25  0.4 1.6 0.02 1.2 60 0.03  2.3 77 5Br 0.006 0.6 100 0.007 0.5 71 0.007 0.6 86 0.007 143

Example 12 Distribution of 5-fluoro-indole Acetic Acid in vivo in Micewith the Carcinoma NT Tumour

5-fluoro-indole acetic acid (5 mg/mL) in 2% v/v ethanol/water wasadjusted to pH 7.4 with NaOH and injected i.p. at a dose of 50 mg/kginto female CBA mice bearing the Carcinoma NT tumour (see example 8 fordetails). The mice were sacrificed up to 2 hours after drugadministration and the blood and tissues removed immediately and placedon ice. The whole blood was spun down and the plasma stored at −20° C.Tissue sample were weighed and homogenised in 4 to 9 volumes of ice-coldwater. The homogenised tissue was then stored at −20° C.

For HPLC analysis, plasma (50 μL) was mixed with IAA internal standard(130 μM, 25 μL) and protein precipitated with acetonitrile (50 μL). Thesamples were spun down and the supernatant injected directly for HPLCanalysis. For tissue levels, samples (250 μL) was mixed with IAAinternal standard (130 μM, 25 μL) and precipitated with acetonitrile(250 μL) for direct injection for HPLC analysis. HPLC analysis wascarried out with a Hypersil SODS 125×4.6 mm column eluting with A; 75%acetonitrile and B: 20 mM ammonium acetate (pH 5.1) with a gradient of15-70% A in 10 minutes at 2 mL/minute. Detection was at 290 nm using aWaters 486 variable wavelength detector.

The amount of 5-fluoro-indole acetic acid in the samples is illustratedin FIG. 6. These results show that sufficient concentrations to achievea cytotoxic effect are attained in the tumour. High levels in the kidneyare consistent with excretion of substantial amounts of the compoundunchanged, which, without wishing to be bound by theory, results fromthe blocking of hydroxylation by P450s by the 5 fluorine substituent.

FIGURE LEGENDS

FIG. 1: -●- with HRP; -∘- Without HRP

FIG. 3: -∘- MCF7 controls; -●- MCF7+5-F-IAA/HRP;

-   -   -□- HT29 controls; -▪- HT29+5-F-IAA/HRP

FIG. 4 a: -∘- methylene blue alone

-   -   -●- methylene blue+5-F-IAA    -   -▴- methylene blue+5-Br-IAA

FIG. 4 b: -∘- thionine alone

-   -   -●- thionine+5-F-IAA    -   -▴- thionine blue+5-Br-IAA

FIG. 4 c: -∘- toluidine blue alone

-   -   -●- toluidine blue+5-F-IAA    -   -▴- toluidine blue+5-Br-IAA

FIG. 4 d: -∘- rose bengal alone

-   -   -●- rose bengal+5-F-IAA    -   -▴- rose bengal+5-Br-IAA

FIG. 5: -∘- control+5-F-IAA;

-   -   -●- transfectants+5-F-IAA;    -   -□- control+5-Br-IAA;    -   -▪- transfectants+5-Br-IAA

FIG. 6: -∘- plasma

-   -   -●- tumour    -   -□- liver    -   ▪- heart    -   -Δ- kidney    -   -▴- muscle

1. A pharmaceutical composition comprising a compound, of formula (I):

and a pharmaceutically acceptable carrier or diluent; wherein R₂ is H;R₁, R₃ and R′₃ are independently selected form H or lower alkyl; R₄, R₅,R₆ and R₇ are independently selected from H, electron withdrawinggroups, lower alkyl groups, lower alkoxy groups, aryl groups or aryloxygroups, wherein at least one of R₄, R₅, R₆ and R₇ is selected from anelectron withdrawing group.
 2. The pharmaceutical composition accordingto claim 1, wherein the electron withdrawing group is selected from thegroup consisting of F, Cl, Br, 1, OCF₃, carboxyl, acetal and electrondeficient aryl.
 3. The pharmaceutical composition according to claim 2,wherein the electron withdrawing group is selected from the groupconsisting of F, Ca, Br and I.
 4. The pharmaceutical compositionaccording to claim 1, wherein the balance of the substituents R₄, R₅, R₆and R₇ is electron withdrawing.
 5. The pharmaceutical compositionaccording to claim 1, wherein R₁ is independently selected from H oroptionally substituted saturated lower alkyl groups.
 6. Thepharmaceutical composition according to claim 5, wherein R₁ isindependently selected from H, methyl or ethyl.
 7. The Pharmaceuticalcomposition according to claim 1, wherein R′₃ is H.
 8. Thepharmaceutical composition according to claim 1, wherein R₃ is selectedfrom H or optionally substituted saturated lower alkyl groups.
 9. Thepharmaceutical composition according to claim 8, wherein R₃, is selectedfrom H, methyl or ethyl.
 10. The pharmaceutical composition according toclaim 1, wherein one or two of R₄, R₅, R₆, and R₇, are independentlyselected from electron withdrawing groups.
 11. The pharmaceuticalcomposition according to claim 1, wherein if one or more of R₄, R₅, R₆,and R₇, are not H or an electron withdrawing groups, they am selectedfrom optionally substituted saturated lower alkyl groups.
 12. Thepharmaceutical composition according to claim 11, wherein those of R₄,R₅, R₆, and R₇, which are not H or an electron withdrawing group areselected from H, methyl or ethyl.
 13. The pharmaceutical compositionaccording to claim 1, wherein one of R₄, R₅, R₆ and R₇, is an electronwithdrawing group and the rest are H.
 14. The pharmaceutical compositionaccording to claim 1, wherein the balance of the substituents R₄, R₆ andR₇, is electron withdrawing.